title: Identification of the binding site for a low-molecular-weight inhibitor of plasminogen activator inhibitor type 1 by site-directed mutagenesis.
abstract: A novel low-molecular-weight inhibitor, AR-H029953XX, was developed from a known fibrinolytic compound, flufenamic acid, which prevented complex formation of human plasminogen activator inhibitor type 1 (PAI-1) with tissue plasminogen activator (tPA) by inhibition of PAI-1. To explore the binding site for AR-H029953XX, mutants of human PAI-1 were constructed by site-directed mutagenesis and were then expressed in CHO cells, purified, activated, and characterized. (1) PAI-1 with mutations in the reactive center loop: L1-PAI-1 (P10, Ser337Glu) had stability and activity similar to those of wild-type PAI-1 (wt-PAI-1), and L2-PAI-1 (P12, Ala335Glu) was highly stable but was a substrate for tPA. (2) PAI-1 with mutations near the binding epitope for the strongly inhibiting monoclonal antibody CLB-2C8: C1-PAI-1 (Phe114Glu), C2-PAI-1 (Val121Phe), C3-PAI-1 (Arg76Glu/Arg115Glu/Arg118Glu), and C4-PAI-1 (Arg115Glu) were all comparable in activity and stability to wt-PAI-1. AR-H029953XX (Ki = 25 microM) prevented complex formation between tPA and active wt-PAI-1 as well as that with mutants L1-, L2-, C1-, C2-, and C4-PAI-1. AR-H029953XX also inhibited binding of these PAI-1 variants to the antibody CLB-2C8, as measured by surface plasmon resonance. In contrast, AR-H029953XX had almost no inhibitory effect on the complex formation of tPA with C3-PAI-1. Moreover, AR-H029953XX had no effect on the binding rate of CLB-2C8 to C3-PAI-1, or on the binding to latent PAI-1 or to cleaved L2-PAI-1. The binding site of AR-H029953XX thus appears to be located in the neighborhood of the postulated epitope for CLB-2C8, near residues Arg76 and/or Arg118. This specific domain of the PAI-1 molecule might thus also be important for the mechanism of inhibitory activity toward tPA. Moreover, the structure of this region in active PAI-1 has to be different from the corresponding regions in latent and cleaved PAI-1.
authors.affiliation: Preclinical R&D, Astra Hässle, Mölndal, Sweden.
authors.forename: P
authors.initials: P
authors.lastname: Björquist
authors.forename: J
authors.initials: J
authors.lastname: Ehnebom
authors.forename: T
authors.initials: T
authors.lastname: Inghardt
authors.forename: L
authors.initials: L
authors.lastname: Hansson
authors.forename: M
authors.initials: M
authors.lastname: Lindberg
authors.forename: M
authors.initials: M
authors.lastname: Linschoten
authors.forename: M
authors.initials: M
authors.lastname: Strömqvist
authors.forename: J
authors.initials: J
authors.lastname: Deinum
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D001666:Binding Sites, Antibody; D015374:Biosensing Techniques; D016466:CHO Cells; D002623:Chemistry Techniques, Analytical; D002742:Chlorphenamidine / Q000378:metabolism*; D002863:Chromogenic Compounds; D006224:Cricetinae; D005439:Flufenamic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009574:Nitro Compounds / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015849:Plasminogen Inactivators / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000378:metabolism
chemical_list: C110601:AR-H029953XX; D000911:Antibodies, Monoclonal; D002863:Chromogenic Compounds; D009574:Nitro Compounds; D017395:Plasminogen Activator Inhibitor 1; D015849:Plasminogen Inactivators; D005439:Flufenamic Acid; D010959:Tissue Plasminogen Activator; D002742:Chlorphenamidine